Publications by authors named "Bart Panis"

46 Publications

Seed Banks as Incidental Fungi Banks: Fungal Endophyte Diversity in Stored Seeds of Banana Wild Relatives.

Front Microbiol 2021 22;12:643731. Epub 2021 Mar 22.

Department of Comparative Plant and Fungal Biology, Royal Botanic Gardens, Kew, Richmond, United Kingdom.

Seed banks were first established to conserve crop genetic diversity, but seed banking has more recently been extended to wild plants, particularly crop wild relatives (CWRs) (e.g., by the Millennium Seed Bank (MSB), Royal Botanic Gardens Kew). CWRs have been recognised as potential reservoirs of beneficial traits for our domesticated crops, and with mounting evidence of the importance of the microbiome to organismal health, it follows that the microbial communities of wild relatives could also be a valuable resource for crop resilience to environmental and pathogenic threats. Endophytic fungi reside asymptomatically inside all plant tissues and have been found to confer advantages to their plant host. Preserving the natural microbial diversity of plants could therefore represent an important secondary conservation role of seed banks. At the same time, species that are reported as endophytes may also be latent pathogens. We explored the potential of the MSB as an incidental fungal endophyte bank by assessing diversity of fungi inside stored seeds. Using banana CWRs in the genus as a case-study, we sequenced an extended ITS-LSU fragment in order to delimit operational taxonomic units (OTUs) and used a similarity and phylogenetics approach for classification. Fungi were successfully detected inside just under one third of the seeds, with a few genera accounting for most of the OTUs-primarily , , and -while a large variety of rare OTUs from across the Ascomycota were isolated only once. species were notably abundant-of significance in light of Fusarium wilt, a disease threatening global banana crops-and so were targeted for additional sequencing with the marker in order to delimit species and place them in a phylogeny of the genus. Endophyte community composition, diversity and abundance was significantly different across habitats, and we explored the relationship between community differences and seed germination/viability. Our results show that there is a previously neglected invisible fungal dimension to seed banking that could well have implications for the seed collection and storage procedures, and that collections such as the MSB are indeed a novel source of potentially useful fungal strains.
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http://dx.doi.org/10.3389/fmicb.2021.643731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024981PMC
March 2021

Challenges and Prospects for the Conservation of Crop Genetic Resources in Field Genebanks, in In Vitro Collections and/or in Liquid Nitrogen.

Plants (Basel) 2020 Nov 24;9(12). Epub 2020 Nov 24.

Alliance of Bioversity International and CIAT, c/o KU Leuven, Willem de Croylaan 42, P.O. Box 2455, 3001 Leuven, Belgium.

The conservation of crop genetic resources, including their wild relatives, is of utmost importance for the future of mankind. Most crops produce orthodox seeds and can, therefore, be stored in seed genebanks. However, this is not an option for crops and species that produce recalcitrant (non-storable) seeds such as cacao, coffee and avocado, for crops that do not produce seeds at all; therefore, they are inevitably vegetatively propagated such as bananas, or crops that are predominantly clonally propagated as their seeds are not true to type, such as potato, cassava and many fruit trees. Field, in vitro and cryopreserved collections provide an alternative in such cases. In this paper, an overview is given on how to manage and setup a field, in vitro and cryopreserved collections, as well as advantages and associated problems taking into account the practical, financial and safety issues in the long-term. In addition, the need for identification of unique accessions and elimination of duplicates is discussed. The different conservation methods are illustrated with practical examples and experiences from national and international genebanks. Finally, the importance of establishing safe and long-term conservation methods and associated backup possibilities is highlighted in the frame of the global COVID-19 pandemic.
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http://dx.doi.org/10.3390/plants9121634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761154PMC
November 2020

Ecological divergence of wild strawberry DNA methylation patterns at distinct spatial scales.

Mol Ecol 2020 12 2;29(24):4871-4881. Epub 2020 Nov 2.

Plant Conservation and Population Biology, University of Leuven, Leuven, Belgium.

Epigenetic change is considered relatively unstable and short-lived, raising questions of its contribution to long-term adaptive potential. However, epigenetic modifications can accumulate in the presence of environmental stress, resulting in beneficial epigenetic memories where environments are challenging. Diverging epigenetic memories have been observed across large spatial scales, and can persist through multiple generations. It is unknown, however, to what extent epigenetic variation contributes to fine-scale population structure and evolution. We compared DNA methylation patterns between a steep, altitudinal gradient (<2 km) and a wide spatial gradient (>500 km) using whole genome bisulphite sequencing data from 30 Fragaria vesca plants germinated and grown in controlled conditions. To assess the stability of spatial epigenetic variation in the presence of an environmental stressor, we applied acute drought stress to part of the plants and quantified drought-induced changes in DNA methylation signatures. We find that epigenetic memories and genomic islands of epigenetic divergence arise even at fine spatial scale, and that distinct spatial scales are featured by distinct epigenetic patterns. For example, demethylation of transposable elements consistently occurred at the large but not the fine spatial scale, while methylation differentiation for most biological processes were shared between spatial scales. Acute drought stress did not result in significant epigenetic differentiation. Our results indicate that population history, rather than short-term environmental stress, plays a dominant role in shaping epigenetic signatures. Specifically, repeated historical stress levels associated with heterogeneous environmental conditions may be required for acquiring a stable epigenetic memory and for coping with future environmental change.
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http://dx.doi.org/10.1111/mec.15689DOI Listing
December 2020

Challenges for Ex Situ Conservation of Wild Bananas: Seeds Collected in Papua New Guinea Have Variable Levels of Desiccation Tolerance.

Plants (Basel) 2020 Sep 21;9(9). Epub 2020 Sep 21.

Department of Biosystems, Katholieke Universiteit Leuven, Willem de Croylaan 42, 3001 Leuven, Belgium.

Ex situ seed conservation of banana crop wild relatives ( spp. L.), is constrained by critical knowledge gaps in their storage and germination behaviour. Additionally, challenges in collecting seeds from wild populations impact the quality of seed collections. It is, therefore, crucial to evaluate the viability of seeds from such collecting missions in order to improve the value of future seed collections. We evaluate the seed viability of 37 accessions of seven species, collected from wild populations in Papua New Guinea, during two collecting missions. Seeds from one mission had already been stored in conventional storage (dried for four months at 15% relative humidity, 20 °C and stored for two months at 15% relative humdity, -20 °C), so a post-storage test was carried out. Seeds from the second mission were assessed freshly extracted and following desiccation. We used embryo rescue techniques to overcome the barrier of germinating in vivo seeds. Seeds from the first mission had low viability (19 ± 27% mean and standard deviation) after storage for two months at 15% relative humidity and -20 °C. Colla seeds had significantly higher post-storage germination than other species ( < 0.01). Desiccation reduced germination of the seeds from the second collecting mission, from 84 ± 22% (at 16.7 ± 2.4% moisture content) to 36 ± 30% (at 2.4 ± 0.8% moisture content). There was considerable variation between and (to a lesser extent) within accessions, a proportion of individual seeds of all but one species ( N.W.Simmonds) survived desiccation and sub-zero temperature storage. We identified that seeds from the basal end of the infructescence were less likely to be viable after storage ( < 0.001); and made morphological observations that identify seeds and infructescences with higher viability in relation to their developmental maturity. We highlight the need for research into seed eco-physiology of crop wild relatives in order to improve future collecting missions.
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http://dx.doi.org/10.3390/plants9091243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570212PMC
September 2020

Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources.

Sci Rep 2020 09 7;10(1):14674. Epub 2020 Sep 7.

Dept. Biosystems, Laboratory of Tropical Crop Improvement, KU Leuven, 3001, Leuven, Belgium.

Sweet potato (Ipomoea batatas) is one of the ten most important staple crops and provides a livelihood for many people around the globe. To adapt to ever-changing circumstances farmers and breeders need to have access to a broad diversity of germplasm. This study focuses on the development of a cryopreservation protocol that allows the long term storage of different sweet potato cultivars. For this, a droplet vitrification protocol was optimized, comparing several parameters; preculture method (0.3 M sucrose vs no preculture); meristem position (axillary vs apical); plant age (3 to 9 weeks); regeneration medium (MS + 2.22 µM BA, Hirai and MS); and length of loading solution treatment (20 to 360 min). Two months after cryopreservation, the regeneration rates of the meristems were compared, which resulted in significant differences for the preculture method, meristem position and loading solution. With these new insights an optimized droplet vitrification protocol was developed with the following parameters: use of 3-9 week old axillary meristems, no preculture phase, 20 min LS treatment, 30 min PVS2 treatment, exposure to liquid nitrogen by droplet vitrification, warming treatment in RS for 15 min, 1 day 0.3 M sucrose recuperation culture, 1 month MS + 2.22 µM BA followed by 1 month of MS cultures. This protocol was subsequently tested on 10 representative accessions resulting in a post cryopreservation regeneration rate of more than 40% for 70% of the tested cultivars, showing that this protocol could be implemented for a large portion of existing sweet potato collections.
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http://dx.doi.org/10.1038/s41598-020-70869-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7477159PMC
September 2020

Is the bacterial leaf nodule symbiosis obligate for Psychotria umbellata? The development of a Burkholderia-free host plant.

PLoS One 2019 16;14(7):e0219863. Epub 2019 Jul 16.

Natural History Museum, University of Oslo, Oslo, Norway.

Background & Aims: The bacterial leaf nodule symbiosis is an interaction where bacteria are housed in specialised structures in the leaves of their plant host. In the Rubiaceae plant family, host plants interact with Burkholderia bacteria. This interaction might play a role in the host plant defence system. It is unique due to its high specificity; the vertical transmission of the endophyte to the next generation of the host plant; and its supposedly obligatory character. Although previous attempts have been made to investigate this obligatory character by developing Burkholderia-free plants, none have succeeded and nodulating plants were still produced. In order to investigate the obligatory character of this endosymbiosis, our aims were to develop Burkholderia-free Psychotria umbellata plants and to investigate the effect of the absence of the endophytes on the host in a controlled environment.

Methods: The Burkholderia-free plants were obtained via embryo culture, a plant cultivation technique. In order to analyse the endophyte-free status, we screened the plants morphologically, microscopically and molecularly over a period of three years. To characterise the phenotype and growth of the in vitro aposymbiotic plants, we compared the growth of the Burkholderia-free plants to the nodulating plants under the same in vitro conditions.

Key Results: All the developed plants were Burkholderia-free and survived in a sterile in vitro environment. The growth analysis showed that plants without endophytes had a slower development.

Conclusions: Embryo culture is a cultivation technique with a high success rate for the development of Burkholderia-free plants of P. umbellata. The increased growth rate in vitro when the specific endophyte is present cannot be explained by possible benefits put forward in previous studies. This might indicate that the benefits of the endosymbiosis are not yet completely understood.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219863PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634412PMC
March 2020

The cryoprotectant PVS2 plays a crucial role in germinating Passiflora ligularis embryos after cryopreservation by influencing the mobilization of lipids and the antioxidant metabolism.

J Plant Physiol 2019 Aug 4;239:71-82. Epub 2019 Jun 4.

Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Katholieke Universiteit Leuven (KU Leuven), W. De Croylaan 42, 3001 Heverlee, Belgium; Bioversity International, W. De Croylaan 42, 3001 Heverlee, Belgium.

Cryopreservation is a process whereby biological structures are preserved in liquid nitrogen (-196 °C) without losing their viability. Many cryopreservation techniques use the Plant Vitrification Solution 2 (PVS2) for cryoprotection. This study will therefore evaluate the influence of different exposure times to the cryoprotectant PVS2 and discuss the importance of the mobilization of reserves and the antioxidant metabolism during the germination of cryopreserved Passiflora ligularis embryos. The composition of P. ligularis seeds was analytically determined. We tested the germination capacity and the Germination Speed Index (GSI) of embryos (that is, seeds without external tegument) which were exposed to different PVS2 exposure times (0, 30, 60 and 120 min) at 30 days after thawing. Proline content, hydrogen peroxide, activity of isocitrate lyase (ICL), malate synthase (MSy), lipid peroxidation and antioxidant enzyme activities (SOD, CAT, APX) were measured at 7, 14 and 21 days after cryopreservation. The germination from cryopreserved embryos was maximal (85%) after 60 min PVS2 exposure with a GSI of 0.6. At 60 min, the highest activity of the enzymes involved in the glyoxylate cycle, ICL and MSy were recorded. We hypothesize that a 60 min exposure to PVS2 accelerates the reserve mobilization which correlates positively with germination. Until 60 min, there was a positive correlation between the PVS2 exposure time and the proline content, as well as the activity of antioxidant enzymes (SOD, CAT, APX), and a negative correlation with the lipid peroxidation. This study enables us to optimize the long-term conservation of this species. In conclusion, fundamental research is necessary to optimize the cryopreservation procedure, and this study offers an effective and efficient workflow which can be extrapolated to other (oil-rich) species.
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http://dx.doi.org/10.1016/j.jplph.2019.05.014DOI Listing
August 2019

Detection of Burkholderia in the seeds of Psychotria punctata (Rubiaceae) - Microscopic evidence for vertical transmission in the leaf nodule symbiosis.

PLoS One 2018 14;13(12):e0209091. Epub 2018 Dec 14.

Natural History Museum, University of Oslo, Oslo, Norway.

Background And Aims: The bacterial leaf nodule symbiosis is a close interaction between endophytes and their plant hosts, mainly within the coffee family. The interaction between Rubiaceae species and Burkholderia bacteria is unique due to its obligate nature, high specificity, and predominantly vertical transmission of the endophytes to the next generation of host plants. This vertical transmission is intriguing since it is the basis for the uniqueness of the symbiosis. However, unequivocal evidence of the location of the endophytes in the seeds is lacking. The aim of this paper is therefore to demonstrate the presence of the host specific endophyte in the seeds of Psychotria punctata and confirm its precise location. In addition, the suggested location of the endophyte in other parts of the host plant is investigated.

Methods: To identify and locate the endophyte in Psychotria punctata, a two-level approach was adopted using both a molecular screening method and fluorescent in situ hybridisation microscopy.

Key Results: The endophytes, molecularly identified as Candidatus Burkholderia kirkii, were detected in the leaves, vegetative and flower buds, anthers, gynoecium, embryos, and young twigs. In addition, they were in situ localised in leaves, flowers and shoot apical meristems, and, for the first time, in between the cotyledons of the embryos.

Conclusions: Both independent techniques detected the host specific endophyte in close proximity to the shoot apical meristem of the embryo, which confirms for the first time the exact location of the endophytes in the seeds. This study provides reliable proof that the endophytes are maintained throughout the growth and development of the host plant and are transmitted vertically to the offspring.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209091PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294375PMC
May 2019

Somatic Embryogenesis in Coffee: The Evolution of Biotechnology and the Integration of Omics Technologies Offer Great Opportunities.

Front Plant Sci 2017 21;8:1460. Epub 2017 Aug 21.

Department of Biosystems, KU LeuvenLeuven, Belgium.

One of the most important crops cultivated around the world is coffee. There are two main cultivated species, and Both species are difficult to improve through conventional breeding, taking at least 20 years to produce a new cultivar. Biotechnological tools such as genetic transformation, micropropagation and somatic embryogenesis (SE) have been extensively studied in order to provide practical results for coffee improvement. While genetic transformation got many attention in the past and is booming with the CRISPR technology, micropropagation and SE are still the major bottle neck and urgently need more attention. The methodologies to induce SE and the further development of the embryos are genotype-dependent, what leads to an almost empirical development of specific protocols for each cultivar or clone. This is a serious limitation and excludes a general comprehensive understanding of the process as a whole. The aim of this review is to provide an overview of which achievements and molecular insights have been gained in (coffee) somatic embryogenesis and encourage researchers to invest further in the technology and combine it with the latest omics techniques (genomics, transcriptomics, proteomics, metabolomics, and phenomics). We conclude that the evolution of biotechnology and the integration of omics technologies offer great opportunities to (i) optimize the production process of SE and the subsequent conversion into rooted plantlets and (ii) to screen for possible somaclonal variation. However, currently the usage of the latest biotechnology did not pass the stage beyond proof of potential and needs to further improve.
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http://dx.doi.org/10.3389/fpls.2017.01460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566963PMC
August 2017

In Vitro Cryopreservation of Date Palm Caulogenic Meristems.

Methods Mol Biol 2017 ;1638:39-48

Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven (K.U. Leuven), Leuven, Belgium.

Cryopreservation is the technology of choice not only for plant genetic resource preservation but also for virus eradication and for the efficient management of large-scale micropropagation. In this chapter, we describe three cryopreservation protocols (standard vitrification, droplet vitrification, and encapsulation vitrification) for date palm highly proliferating meristems that are initiated from vitro-cultures using plant growth regulator-free MS medium. The positive impact of sucrose preculture and cold hardening treatments on survival rates is significant. Regeneration rates obtained with standard vitrification, encapsulation-vitrification, and droplet-vitrification protocols can reach 30, 40, and 70%, respectively. All regenerated plants from non-cryopreserved or cryopreserved explants don't show morphological variation by maintaining genetic integrity without adverse effect of cryogenic treatment. Cryopreservation of date palm vitro-cultures enables commercial tissue culture laboratories to move to large-scale propagation from cryopreserved cell lines producing true-to-type plants after clonal field-testing trials. When comparing the cost of cryostorage and in-field conservation of date palm cultivars, tissue cryopreservation is the most cost-effective. Moreover, many of the risks linked to field conservation like erosion due to climatic, edaphic, and phytopathologic constraints are circumvented.
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http://dx.doi.org/10.1007/978-1-4939-7159-6_4DOI Listing
April 2018

Elucidation of the compatible interaction between banana and Meloidogyne incognita via high-throughput proteome profiling.

PLoS One 2017 2;12(6):e0178438. Epub 2017 Jun 2.

Genetics and Molecular Biology Division, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia.

With a diverse host range, Meloidogyne incognita (root-knot nematode) is listed as one of the most economically important obligate parasites of agriculture. This nematode species establishes permanent feeding sites in plant root systems soon after infestation. A compatible host-nematode interaction triggers a cascade of morphological and physiological process disruptions of the host, leading to pathogenesis. Such disruption is reflected by altered gene expression in affected cells, detectable using molecular approaches. We employed a high-throughput proteomics approach to elucidate the events involved in a compatible banana- M. incognita interaction. This study serves as the first crucial step in developing natural banana resistance for the purpose of biological-based nematode management programme. We successfully profiled 114 Grand naine root proteins involved in the interaction with M. incognita at the 30th- and 60th- day after inoculation (dai). The abundance of proteins involved in fundamental biological processes, cellular component organisation and stress responses were significantly altered in inoculated root samples. In addition, the abundance of proteins in pathways associated with defence and giant cell maintenance in plants such as phenylpropanoid biosynthesis, glycolysis and citrate cycle were also implicated by the infestation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178438PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5456091PMC
October 2017

Direct nematicidal effects of methyl jasmonate and acibenzolar-S-methyl against Meloidogyne incognita.

Nat Prod Res 2017 May 23;31(10):1219-1222. Epub 2016 Sep 23.

a Laboratory of Tropical Crop Improvement, Department of Biosystems , University of Leuven (KU Leuven) , Heverlee , Belgium.

The aim of this study was to examine the nematicidal properties of two defence inducers against the root-knot nematode Meloidogyne incognita. A direct-contact bioassay was applied to evaluate the nematicidal effects of acibenzolar-S-methyl (ASM) and methyl jasmonate (MEJA) on second-stage juveniles (J2). Nematodes were incubated in different concentrations of these compounds, and the numbers of immobile nematodes were counted after 24 and 48 h post incubation. Tap water was then added to verify whether the nematodes recovered or remained dead at 72 h. The percentage of dead nematodes was used as indicator for the toxicity of the different solutions. Our results show that ASM, in the formulation of Bion®, and MEJA have nematicidal properties.
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http://dx.doi.org/10.1080/14786419.2016.1230111DOI Listing
May 2017

The proteome profile of embryogenic cell suspensions of Coffea arabica L.

Proteomics 2016 Mar 1;16(6):1001-5. Epub 2016 Mar 1.

Biosystems Department, KULeuven, Leuven, Belgium.

Somatic embryogenesis, is a process by which new viable embryos are produced from somatic tissues. Somatic embryogenesis is not only a useful biotechnological tool for the massive clonal propagation and genetic engineering but it also allows to obtain fundamental knowledge about the molecular changes that take place during embryogenesis. We present the proteome profile of two embryogenic cell suspensions. We identified 1052 non-redundant proteins. We present their known GO annotations and show two protein networks sharing the GO annotations related to stress and embryogenic capacity via the free program Cytoscape. To our knowledge these results give the first high-throughput proteome description of embryogenic cell suspensions and provide new information about somatic embryos for the whole plant community. The published proteome is a first step toward understanding somatic embryogenesis in coffee and toward a better annotation of proteins in an important non-model crop. All data are available via ProteomeXchange with identifier PXD002963.
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http://dx.doi.org/10.1002/pmic.201500399DOI Listing
March 2016

Arbuscular Mycorrhizal Fungi for the Biocontrol of Plant-Parasitic Nematodes: A Review of the Mechanisms Involved.

Front Microbiol 2015 17;6:1280. Epub 2015 Nov 17.

Centre of Microbial and Plant Genetics , KU Leuven, Heverlee, Belgium ; Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie , Gent, Belgium ; Commonwealth Scientific and Industrial Research Organisation Agriculture, Queensland Bioscience Precinct , Brisbane, QLD, Australia.

Arbuscular mycorrhizal fungi (AMF) are obligate root symbionts that can protect their host plant against biotic stress factors such as plant-parasitic nematode (PPN) infection. PPN consist of a wide range of species with different life styles that can cause major damage in many important crops worldwide. Various mechanisms have been proposed to play a role in the biocontrol effect of AMF against PPN. This review presents an overview of the different mechanisms that have been proposed, and discusses into more detail the plausibility of their involvement in the biocontrol against PPN specifically. The proposed mechanisms include enhanced plant tolerance, direct competition for nutrients and space, induced systemic resistance (ISR) and altered rhizosphere interactions. Recent studies have emphasized the importance of ISR in biocontrol and are increasingly placing rhizosphere effects on the foreground as well, both of which will be the focal point of this review. Though AMF are not yet widely used in conventional agriculture, recent data help to develop a better insight into the modes of action, which will eventually lead toward future field applications of AMF against PPN. The scientific community has entered an exciting era that provides the tools to actually unravel the underlying molecular mechanisms, making this a timely opportunity for a review of our current knowledge and the challenges ahead.
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http://dx.doi.org/10.3389/fmicb.2015.01280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646980PMC
December 2015

Unravelling the effect of sucrose and cold pretreatment on cryopreservation of potato through sugar analysis and proteomics.

Cryobiology 2015 Dec 25;71(3):432-41. Epub 2015 Sep 25.

Environment Research and Innovation Department (ERIN), Luxembourg Institute of Science and Technology, 5, Avenue des Hauts-Fourneaux, L-4362 Esch/Alzette, GD, Luxembourg.

Apical shoot tips were dissected from donor plants (cultured in several conditions) and cryopreserved using the droplet-vitrification technique. The effect of two preculture treatments (sucrose pretreatment medium or cold-culturing during two weeks) on donor plants of four potato species (Solanum commersonii, S. juzepcukii, S. ajanhuiri, and Solanum tuberosum) was studied. Post-cryopreservation meristem growth and plant recovery were influenced by the treatments, but the effect on the regeneration was strongly genotype-dependent. The highest post-rewarming plant recovery percentage was obtained using meristems dissected from donor plants of S. commersonii cultured on sucrose pretreatment medium or cold-cultured. Both preculture conditions also enhanced plant recovery in S. juzepcukii compared to control cultures. Cold preculture, however, proved to be undesirable for S. tuberosum whereas sucrose pretreatment had a positive impact on the plant regeneration of this species. The determination of changes in the concentration of soluble sugars revealed sugar accumulation, especially of sucrose and the raffinose family of oligosaccharides (RFOs), which can be linked to tolerance towards the cryopreservation. Additionally, a study of the proteome of the donor plantlets after the pretreatments by 2D-fluorescence difference gel electrophoresis (DIGE) was carried out to identify differentially abundant proteins. Carbon metabolism-related proteins, together with stress-response and oxidative-homeostasis related proteins were the main class of proteins that changed in abundance after the pretreatments. Our results suggest that oxidative homeostasis-related proteins and sugars may be associated with the improved tolerance to cryopreservation and the ability to cold acclimate by S. commersonii in contrast to the other genotypes. The increased accumulation of sucrose and RFOs play a fundamental role in the response to stress in potato and may help to acquire tolerance to cryopreservation.
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http://dx.doi.org/10.1016/j.cryobiol.2015.09.006DOI Listing
December 2015

Cryopreservation of Bituminaria bituminosa varieties and hybrids.

Cryobiology 2015 Oct 29;71(2):279-85. Epub 2015 Jul 29.

Bioversity International, Willem de Croylaan 42, 3001 Leuven, Belgium. Electronic address:

Bituminaria bituminosa (L.) C.H. Stirton is a drought tolerant, perennial legume pasture species and a source of pharmaceutical compounds. Bituminaria breeding programs aim to develop and conserve hybrids with desirable traits such as high forage quality, tolerance to biotic or abiotic stresses, and high contents of furanocoumarins. In this work we present a cryopreservation study of different B. bituminosa accessions: two varieties and eight intervarietal hybrids resulting from crosses between the three botanical varieties: var. bituminosa, var. crassiuscula, and var. albomarginata. No previous work on cryopreservation of Bituminaria species has been reported. We applied the ultra-fast cooling method, using droplet vitrification on aluminum foil strips. First, we investigated the PVS2 toxicity and cryopreservation damage in two genotypes, comparing three PVS2 treatments and two culture media. An incubation of 30 min in PVS2 resulted in regeneration rates after cryopreservation higher than 80%. The MS medium was selected for optimal meristem outgrowth, in order to avoid the prominent callus formation that was observed in the presence of BAP. These conditions were subsequently used to cryopreserve eight other genotypes. The results were highly variable; 45 days after cryopreservation, survival ranged between 22% and 98% while regeneration ranged between 0% and 96%, depending on the accession. A significant and positive correlation was observed between survival and regeneration. At 90 days post culture plantlets could be recovered from cryopreserved explants of all genotypes. This study shows that the droplet vitrification method is promising for the cryopreservation of eight of the 10 genotypes assayed and the method can thus be applied to develop a cryobank of B. bituminosa.
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http://dx.doi.org/10.1016/j.cryobiol.2015.07.011DOI Listing
October 2015

Cryopreservation of Neottopteris nidus prothallus and green globular bodies by droplet-vitrification.

Cryo Letters 2013 Sep-Oct;34(5):481-9

Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Department of Biosystems, K. U. Leuven, B3001 Leuven, Belgium.

Neottopteris nidus prothalli and green globular bodies (GGBs) were successfully cryopreserved by droplet-vitrification. Prothalli were subjected to different treatments. The following parameters were studied: the age of in vitro mother plants from which prothalli were originated (30 to 90-day old), length of exposure to loading solution (LS) (20 to 40 min) and length of exposure to the plant vitrification solution (PVS2) (10 to 55 min). N. nidus GGBs and GGBs in suspension were subjected to PVS2 for 20, 30 and 40 min before liquid nitrogen exposure. The highest prothalli regrowth (92%) occurred when they were exposed for 40 min to LS, followed by 20 min to PVS2 and when they originated from non-preconditioned 45-day old mother plants. The highest GGB (100%) and GGB suspension regrowth (100%) after cryopreservation occurred when they were exposed to PVS2 for either 20 min or 40 min.
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February 2014

Changes in sugar content and proteome of potato in response to cold and dehydration stress and their implications for cryopreservation.

J Proteomics 2014 Feb 10;98:99-111. Epub 2013 Dec 10.

Bioversity International, Willem de Croylaan 42 bus 2455, B-3001 Leuven, Belgium.

Unlabelled: The key to successful cryopreservation lies in the induction of tolerance towards dehydration/desiccation and freezing. The accumulation of osmo-active compounds, which can be induced by drought and cold stress, is therefore important. In the present study, three-week old shoots from in vitro plantlets of the cultivated potato Solanum tuberosum and its frost-resistant relative Solanum commersonii were submitted to osmotic stress (by using sucrose) and chilling (6°C). After 14days of exposure, shoot tips were sampled in order to gain an insight into changes of the proteome and soluble sugars. Also, the effect of these treatments on growth performance behaviour and on the success of cryopreservation was evaluated. Identified proteins that changed in abundance due to stress were associated with stress response. Additionally, carbohydrate analyses in both species, after exposure to chilling, also indicated species-related differences; this observation could point towards a better-adapted physiological state of the donor plants of S. commersonii prior to the cryoprocedure and therefore a better recovery of the meristems.

Biological Significance: To our knowledge, this is the first study in which cryopreservation experiments are combined with the observation of the responses to abiotic stress exposure involving the potato species S. commersonii and S. tuberosum. These two species are known to have a different cold-acclimation behaviour, which seems to be closely related to their tolerance towards cryopreservation. Furthermore, common and differential responses to abiotic stresses were observed in the two species indicating that some pathways could be crucial not only in the plant's response to stress but also in tolerance towards cryopreservation.
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http://dx.doi.org/10.1016/j.jprot.2013.11.027DOI Listing
February 2014

The use of 2D-DIGE to understand the regeneration of somatic embryos in avocado.

Proteomics 2013 Dec;13(23-24):3498-507

IFAPA, Centro de Churriana, Málaga, Spain.

Avocado embryogenic cell cultures can be classified into two groups based on their morphology when cultured on a medium containing auxin: somatic embryo (SE) and proembryonic masses (PEM) type cultures. The calli of SE-type cell lines are able to go through the maturation process, whereas the calli of PEM cell lines rarely mature. We have investigated four independent avocado cell cultures (two SE and two PEM). The aim of this study was to link the differential regeneration capacity of the four cell cultures to a proteomic pattern and to gain insight into the regeneration capacity. A 2D-DIGE analysis followed by a blind multivariate analysis was able to separate the two SE lines from the PEM lines indicating that the protein profiles of SE and PEM calli are different. Based on the variable importance, that is, the differential protein pattern, we hypothesize that the regeneration capacity in avocado is correlated to the ability to overcome the physicochemical stress stimuli associated with the in vitro culture. Our identical culture conditions do not seem to trigger an appropriate response in PEM lines.
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http://dx.doi.org/10.1002/pmic.201300148DOI Listing
December 2013

Differential Protein Expression in Response to Abiotic Stress in Two Potato Species: Solanum commersonii Dun and Solanum tuberosum L.

Int J Mol Sci 2013 Mar 1;14(3):4912-33. Epub 2013 Mar 1.

Department Environment and Agro-biotechnologies (EVA), Centre de Recherche Public-Gabriel Lippmann, 41, rue du Brill, L-4422 Belvaux, Luxembourg.

Better knowledge on responses to dehydration stress could help to improve the existing cryopreservation protocols for potato, since plant tissues processed for cryopreservation are often submitted to similar in vitro stress conditions. Cryopreservation (the best method of conservation for vegetatively propagated plants) of potato still needs to be standardized to make it available and to conserve the wide diversity of this crop. In the present work, the response to osmotic stress and chilling temperature was investigated in two potato species, Solanum tuberosum and its relative, frost-tolerant S. commersonii. After 14 days of exposure, different growth parameters, such as shoot length and number of leaves, were measured. Furthermore, differentially abundant proteins were identified after performing 2-fluorescence difference gel electrophoresis (2-DIGE) experiments, and soluble carbohydrates were analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). The results show different responses in both species depending on the stress treatment. Focusing on the differences in growth parameters during the treatments, Solanum commersonii seems to be more affected than S. tuberosum cv. Désirée. At the molecular level, there are some differences and similarities between the two potato species studied that are dependent on the type of stressor.
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http://dx.doi.org/10.3390/ijms14034912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3634427PMC
March 2013

Thermotherapy, chemotherapy, and meristem culture in banana.

Methods Mol Biol 2013 ;11013:419-33

Plant Pathology Unit, University of Liege, Gembloux Agro-Bio Tech, Gembloux, Belgium.

Bananas that provide a staple food to the millions of people are adversely affected by several viruses such as Banana bunchy Top Virus (BBTV), Banana Streak Virus (BSV), and Cucumber Mosaic Virus (CMV). These viruses are known to have a devastating effect on crop production and constraint to the international exchange and conservation of banana germplasm-a cornerstone for breeding new cultivars. The viruses are particularly problematic in vegetative propagated crops, like bananas, because of their transmission in the planting material. Different virus eradication techniques have been developed, such as thermotherapy, chemotherapy, and meristem culture for providing virus-free planting material. Meristem culture proved to be the most effective procedure to eradicate phloem-associated viruses. This method requires isolation of meristematic dome of plant under the aseptic conditions and culture in an appropriate nutrient medium to develop new virus-free plants. Thermotherapy is another widely used virus eradication technique, which is initially carried out on in vivo or in vitro plants and eventually combined with meristem culture technique. The plantlets are initially grown at 28°C day temperature and increase it by 2°C per day until reaches 40°C and the night temperature at 28°C; maintain plants at 40°C for 4 weeks; excise meristem and culture onto the regeneration medium. In chemotherapy technique, antiviral chemical compound Virazole(®) is applied on meristem culture. Combination of these techniques is also applied to improve the eradication rate.
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http://dx.doi.org/10.1007/978-1-62703-074-8_32DOI Listing
May 2013

Screening the banana biodiversity for drought tolerance: can an in vitro growth model and proteomics be used as a tool to discover tolerant varieties and understand homeostasis.

Front Plant Sci 2012 2;3:176. Epub 2012 Aug 2.

Division of Crop Biotechnics, KULeuven Leuven, Belgium.

There is a great need for research aimed at understanding drought tolerance, screening for drought tolerant varieties and breeding crops with an improved water use efficiency. Bananas and plantains are a major staple food and export product with a worldwide production of over 135 million tonnes per year. Water however is the most limiting abiotic factor in banana production. A screening of the Musa biodiversity has not yet been performed. We at KU Leuven host the Musa International Germplasm collection with over 1200 accessions. To screen the Musa biodiversity for drought tolerant varieties, we developed a screening test for in vitro plants. Five varieties representing different genomic constitutions in banana (AAAh, AAA, AAB, AABp, and ABB) were selected and subjected to a mild osmotic stress. The ABB variety showed the smallest stress induced growth reduction. To get an insight into the acclimation and the accomplishment of homeostasis, the leaf proteome of this variety was characterized via 2D DIGE. After extraction of the leaf proteome of six control and six stressed plants, 2600 spots could be distinguished. A PCA analysis indicates that control and stressed plants can blindly be classified based on their proteome. One hundred and twelve proteins were significantly more abundant in the stressed plants and 18 proteins were significantly more abundant in control plants (FDR α 0.05). Twenty four differential proteins could be identified. The proteome analysis clearly shows that there is a new balance in the stressed plants and that the respiration, metabolism of ROS and several dehydrogenases involved in NAD/NADH homeostasis play an important role.
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http://dx.doi.org/10.3389/fpls.2012.00176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410380PMC
August 2012

Palm cryobanking.

Cryo Letters 2011 Nov-Dec;32(6):451-62

Laboratory of Plant Biotechnology, Faculty of Sciences, University of Sfax, Sfax, Tunisia.

We describe the development of an efficient cryopreservation protocol for proembryogenic masses (PEMs) of date palm variety 'Barhee'. Proembryos were induced by inoculating small pieces of juvenile leaves on MS medium supplemented with 0.3 mg per liter 2,4-D. Application of these in vitro conditions led to true-to-type plants as observed after plant fructification. When compared to the standard vitrification protocol, the ultra-rapid droplet-vitrification technique proved to be superior. Sucrose preculture considerably increased post-cryopreservation recovery. The highest regeneration after cryogenic exposure reached 63.3 percent when PEMs were treated with PVS2 for 30 min at 0 degree C and 56.7 percent when PVS2 treatment lasted for 15 min at 25 degree C. The first signs of regrowth of cryopreserved PEMs were observed after 2 to 3 weeks. Cryopreservation did not affect the morphogenetic capacities of the plant material. Moreover, highly proliferating suspension cultures could be established from the cryopreserved material. The overall production of somatic embryos from 500 mg cryopreserved PEMs reached 1030 +/- 50 units after 1 month. The morphological study of date palms regenerated from cryopreserved material confirmed the stability of clonal material following cryopreservation.
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February 2012

Arbuscular mycorrhizal fungi affect both penetration and further life stage development of root-knot nematodes in tomato.

Mycorrhiza 2012 Feb 7;22(2):157-63. Epub 2011 Dec 7.

Laboratory of Tropical Crop Improvement, Department of Biosystems, University of Leuven (K.U. Leuven), Kasteelpark Arenberg 13, Leuven, Belgium.

The root-knot nematode Meloidogyne incognita poses a worldwide threat to agriculture, with an increasing demand for alternative control options since most common nematicides are being withdrawn due to environmental concerns. The biocontrol potential of arbuscular mycorrhizal fungi (AMF) against plant-parasitic nematodes has been demonstrated, but the modes of action remain to be unraveled. In this study, M. incognita penetration of second-stage juveniles at 4, 8 and 12 days after inoculation was compared in tomato roots (Solanum lycopersicum cv. Marmande) pre-colonized or not by the AMF Glomus mosseae. Further life stage development of the juveniles was also observed in both control and mycorrhizal roots at 12 days, 3 weeks and 4 weeks after inoculation by means of acid fuchsin staining. Penetration was significantly lower in mycorrhizal roots, with a reduction up to 32%. Significantly lower numbers of third- and fourth-stage juveniles and females accumulated in mycorrhizal roots, at a slower rate than in control roots. The results show for the first time that G. mosseae continuously suppresses root-knot nematodes throughout their entire early infection phase of root penetration and subsequent life stage development.
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http://dx.doi.org/10.1007/s00572-011-0422-yDOI Listing
February 2012

Plant proteomics in Europe--COST action FA0603.

J Proteomics 2011 Aug 14;74(8):1161-4. Epub 2011 Jul 14.

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http://dx.doi.org/10.1016/j.jprot.2011.07.005DOI Listing
August 2011

Structure and regulation of the Asr gene family in banana.

Planta 2011 Oct 1;234(4):785-98. Epub 2011 Jun 1.

Division of Crop Biotechnics, Laboratory of Tropical Crop Improvement, KULeuven, Kasteelpark Arenberg 13, Bus 2455, 3001 Leuven, Belgium.

Abscisic acid, stress, ripening proteins (ASR) are a family of plant-specific small hydrophilic proteins. Studies in various plant species have highlighted their role in increased resistance to abiotic stress, including drought, but their specific function remains unknown. As a first step toward their potential use in crop improvement, we investigated the structure and regulation of the Asr gene family in Musa species (bananas and plantains). We determined that the Musa Asr gene family contained at least four members, all of which exhibited the typical two exons, one intron structure of Asr genes and the "ABA/WDS" (abscisic acid/water deficit stress) domain characteristic of Asr genes. Phylogenetic analyses determined that the Musa Asr genes were closely related to each other, probably as the product of recent duplication events. For two of the four members, two versions corresponding to the two sub-genomes of Musa, acuminata and balbisiana were identified. Gene expression and protein analyses were performed and Asr expression could be detected in meristem cultures, root, pseudostem, leaf and cormus. In meristem cultures, mAsr1 and mAsr3 were induced by osmotic stress and wounding, while mAsr3 and mAsr4 were induced by exposure to ABA. mASR3 exhibited the most variation both in terms of amino acid sequence and expression pattern, making it the most promising candidate for further functional study and use in crop improvement.
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http://dx.doi.org/10.1007/s00425-011-1421-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3180632PMC
October 2011

Unraveling tobacco BY-2 protein complexes with BN PAGE/LC-MS/MS and clustering methods.

J Proteomics 2011 Aug 2;74(8):1201-17. Epub 2011 Apr 2.

Center for Proteomics (CFP), Groenenborgerlaan 171, B-2020 Antwerp, Belgium.

To understand physiological processes, insight into protein complexes is very important. Through a combination of blue native gel electrophoresis and LC-MS/MS, we were able to isolate protein complexes and identify their potential subunits from Nicotiana tabacum cv. Bright Yellow-2. For this purpose, a bioanalytical approach was used that works without a priori knowledge of the interacting proteins. Different clustering methods (e.g., k-means and hierarchical clustering) and a biclustering approach were evaluated according to their ability to group proteins by their migration profile and to correlate the proteins to a specific complex. The biclustering approach was identified as a very powerful tool for the exploration of protein complexes of whole cell lysates since it allows for the promiscuous nature of proteins. Furthermore, it searches for associations between proteins that co-occur frequently throughout the BN gel, which increases the confidence of the putative associations between co-migrating proteins. The statistical significance and biological relevance of the profile clusters were verified using functional gene ontology annotation. The proof of concept for identifying protein complexes by our BN PAGE/LC-MS/MS approach is provided through the analysis of known protein complexes. Both well characterized long-lived protein complexes as well as potential temporary sequential multi-enzyme complexes were characterized.
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http://dx.doi.org/10.1016/j.jprot.2011.03.023DOI Listing
August 2011

The use of 2D-electrophoresis and de novo sequencing to characterize inter- and intra-cultivar protein polymorphisms in an allopolyploid crop.

Phytochemistry 2011 Jul 23;72(10):1243-50. Epub 2010 Nov 23.

K.U.Leuven, Department of Biosystems, Division of Crop Biotechnics, Leuven, Belgium.

Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops. Several techniques exist to characterize allopolyploid varieties. Analyzing the consequences of genomic reorganization at the gDNA level is a prerequisite but a better insight into the consequences for the phenotype is also primordial. As such, protein polymorphism analysis is important in understanding plant and crop biodiversity and is a driving force behind crop improvement. Our strategy to analyze protein isoforms and to detect possible gene silencing or deletion in bananas was based on protein analysis. Bananas are a good representative of a complex allopolyploid and important crop. We combined two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/MS sequence determination to characterize a range of triploid varieties. Via Principal Component Analysis (PCA) and hierarchical clustering we were able to blindly classify the different varieties according to their presumed genome constitution. We report for the first time the application of an automated approach for the derivatization of peptides for facilitated MS/MS de novo sequence determination. We conclude that the proteome does not always correspond to the presumed genome formulae and that proteomics is a powerful tool to characterize varieties. The observations at the protein level provide good indications for a more complex genome structure and genomic rearrangement in some banana varieties.
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http://dx.doi.org/10.1016/j.phytochem.2010.10.016DOI Listing
July 2011

Recovery and characterisation of hybrid firs (Abies alba x A. cephalonica, Abies albax A. numidica) embryogenic tissues after cryopreservation.

Cryo Letters 2010 May-Jun;31(3):206-17

Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, PO Box 39A, 95007 Nitra, Slovak Republic.

Embryogenic tissues of hybrid firs were cryopreserved using a slow freezing protocol. The procedure involved preculture of tissues for 24, 48 or 72 h in media with different sorbitol concentrations (0.4 or 0.8 M) and addition of 5% (v/v) DMSO as cryoprotectant. The cell lines tested withstood cryopreservation, even though tissue regrowth after thawing was dependent on treatment and cell line. For cell line AN72, regrowth was 100% for all experimental conditions tested. With cell line AC78, regrowth was 100% except after shorter pretreatment durations, which produced 83% and 86% regrowth for 0.4 M and 0.8 M sorbitol pretreatment, respectively. Cell lines AC1 and AC4 were more sensitive to cryopreservation with 37.5 to 100% regrowth, respectively. Growth parameters evaluated 3 months after cryopreservation showed cell line and treatments effects. In most cases, cryopreservation had no negative effect on growth of tissues. Statistically significant differences in fresh mass accumulation were found for four samples out of 24 investigated, although growth increase of these tissues still reached 79.4-84.6%, compared with non-cryopreserved ones (100% increase). Maturation capacity and genetic fidelity were studied in tissues whose growth was not negatively influenced by cryopreservation. Maturation capacity of embryogenic tissues cryopreserved using the optimal protocol was comparable to that of non-frozen controls. RAPD analysis of 88 genomic regions per cell line did not reveal any changes in genetic fidelity of cryopreserved tissues compared to non-cryopreserved controls.
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November 2010

Sugar-mediated acclimation: the importance of sucrose metabolism in meristems.

J Proteome Res 2010 Oct;9(10):5038-46

Division of Crop Biotechnics, K.U. Leuven, Leuven, Belgium.

We have designed an in vitro experimental setup to study the role of sucrose in sugar-mediated acclimation of banana meristems using established highly proliferating meristem cultures. It is a first step toward the systems biology of a meristem and the understanding of how it can survive severe abiotic stress. Using the 2D-DIGE proteomic approach and a meristem-specific EST library, we describe the long-term acclimation response of banana meristems (after 2, 4, 8, and 14 days) and analyze the role of sucrose in this acclimation by setting up a control, a sorbitol, and a sucrose acclimation treatment over time. Sucrose synthase is the dominant enzyme for sucrose breakdown in meristem tissue, which is most likely related to its lower energy consumption. Metabolizing sucrose is of paramount importance to survive, but the uptake of sugar and its metabolism also drive respiration, which may result in limited oxygen levels. According to our data, a successful acclimation is correlated to an initial efficient uptake of sucrose and subsequently a reduced breakdown of sucrose and an induction of fermentation likely by a lack of oxygen.
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http://dx.doi.org/10.1021/pr100321vDOI Listing
October 2010